参考文献

The intestinal stem cell (ISC) marker LGR5 is a receptor for R-spondin (RSPO) that functions to potentiate Wnt signalling in the proliferating crypt. It has been recently shown that Wnt plays a priming role for ISC self-renewal by inducing RSPO receptor LGR5 expression. Despite its pivotal role in homeostasis,regeneration and cancer,little is known about the post-translational regulation of LGR5. Here,we show that the HECT-domain E3 ligases NEDD4 and NEDD4L are expressed in the crypt stem cell regions and regulate ISC priming by degrading LGR receptors. Loss of Nedd4 and Nedd4l enhances ISC proliferation,increases sensitivity to RSPO stimulation and accelerates tumour development in Apcmin mice with increased numbers of high-grade adenomas. Mechanistically,we find that both NEDD4 and NEDD4L negatively regulate Wnt/$\beta$-catenin signalling by targeting LGR5 receptor and DVL2 for proteasomal and lysosomal degradation. Our findings unveil the previously unreported post-translational control of LGR receptors via NEDD4/NEDD4L to regulate ISC priming. Inactivation of NEDD4 and NEDD4L increases Wnt activation and ISC numbers,which subsequently enhances tumour predisposition and progression. View Publication

The network topology of a protein interactome is shaped by the function of each protein,making it a resource of functional knowledge in tissues and in single cells. Today,this resource is underused,as complete network topology characterization has proved difficult for large protein interactomes. We apply a matrix visualization and decoding approach to a physical protein interactome of a dendritic cell,thereby characterizing its topology with no prior assumptions of structure. We discover 294 proteins,each forming topological motifs called bow-ties" that tie together the majority of observed protein complexes. The central proteins of these bow-ties have unique network properties display multifunctional capabilities are enriched for essential proteins and are widely expressed in other cells and tissues. Collectively the bow-tie motifs are a pervasive and previously unnoted topological trend in cellular interactomes. As such these results provide fundamental knowledge on how intracellular protein connectivity is organized and operates." View Publication

Severe acute respiratory syndrome (SARS) coronavirus (CoV) envelope (E) protein is a transmembrane protein. Several subcellular locations and topological conformations of E protein have been proposed. To identify the correct ones,polyclonal and monoclonal antibodies specific for the amino or the carboxy terminus of E protein,respectively,were generated. E protein was mainly found in the endoplasmic reticulum-Golgi intermediate compartment (ERGIC) of cells transfected with a plasmid encoding E protein or infected with SARS-CoV. No evidence of E protein presence in the plasma membrane was found by using immunofluorescence,immunoelectron microscopy and cell surface protein labeling. In addition,measurement of plasma membrane voltage gated ion channel activity by whole-cell patch clamp suggested that E protein was not present in the plasma membrane. A topological conformation in which SARS-CoV E protein amino terminus is oriented towards the lumen of intracellular membranes and carboxy terminus faces cell cytoplasm is proposed. View Publication

Messenger RNA (mRNA) represents an attractive therapeutic modality for potentially a wide range of clinical indications but requires uridine chemistry modification and/or tuning of the production process to prevent activation of cellular innate immune sensors and a concomitant reduction in protein expression. To decipher the relative contributions of these factors on immune activation,here,we compared,in multiple cell and in vivo models,mRNA that encodes human erythropoietin incorporating either canonical uridine or N1-methyl-pseudouridine (1m$\Psi$),synthesized by either a standard process shown to have double-stranded RNA (dsRNA) impurities or a modified process that yields a highly purified mRNA preparation. Our data demonstrate that the lowest stimulation of immune endpoints was with 1m$\Psi$ made by the modified process,while mRNA containing canonical uridine was immunostimulatory regardless of process. These findings confirm that uridine modification and the reduction of dsRNA impurities are both necessary and sufficient at controlling the immune-activating profile of therapeutic mRNA. View Publication

Integrating nutrient sensing with the synthesis of complex molecules is a central feature of metabolism. Yet the regulatory mechanisms underlying such integration are often unknown. Here,we establish that the transcription regulators Rtg1/3 are key determinants of sphingolipid homeostasis in the human fungal pathogen Candida albicans. Quantitative analysis of the C. albicans lipidome reveals Rtg1/3-dependent alterations in all complex sphingolipids and their precursors,ceramides. Mutations in the regulators render the fungus susceptible to myriocin,a sphingolipid synthesis inhibitor. Rtg1/3 exert control on the expression of several enzymes involved in the synthesis of sphingolipids' building blocks,and the regulators are activated upon engulfment of C. albicans cells by human neutrophils. We demonstrate that Rtg1p and Rtg3p are regulated at two levels,one in response to sphingolipids and the other by the nutrient sensor TOR. Our findings,therefore,indicate that the Rtg1/3 system integrates nutrient sensing into the synthesis of complex lipids. View Publication

Periodontitis is an irreversible,bacteria-induced,chronic inflammatory disease that compromises the integrity of tooth-supporting tissues and adversely affects systemic health. As the immune system's first line of defense against bacteria,neutrophils use their microbicidal functions in the oral cavity to protect the host against periodontal disease. However,periodontal pathogens have adapted to resist neutrophil microbicidal mechanisms while still propagating inflammation,which provides essential nutrients for the bacteria to proliferate and cause disease. Advances in sequencing technologies have recognized several newly appreciated bacteria associated with periodontal lesions such as the Gram-positive anaerobic rod,Filifactor alocis. With the discovery of these oral bacterial species,there is also a growing need to assess their pathogenic potential and determine their contribution to disease progression. Currently,few studies have addressed the pathogenic mechanisms used by oral bacteria to manipulate the neutrophil functional responses at the level of the transcriptome. Thus,this study aims to characterize the global changes at the gene expression level in human neutrophils during infection with F. alocis. Our results indicate that the challenge of human neutrophils with F. alocis results in the differential expression of genes involved in multiple neutrophil effector functions such as chemotaxis,cytokine and chemokine signaling pathways,and apoptosis. Moreover,F. alocis challenges affected the expression of components from the TNF and MAPK kinase signaling pathways. This resulted in transient,dampened p38 MAPK activation by secondary stimuli TNF$\alpha$ but not by fMLF. Functionally,the F. alocis-mediated inhibition of p38 activation by TNF$\alpha$ resulted in decreased cytokine production but had no effect on the priming of the respiratory burst response or the delay of apoptosis by TNF$\alpha$. Since the modulatory effect was characteristic of viable F. alocis only,we propose this as one of F. alocis' mechanisms to control neutrophils and their functional responses. View Publication

BACKGROUND: Despite increasing demand,current protocols for human pluripotent stem cell (hPSC)-derived retinal pigment epithelium (RPE) remain time,labor,and cost intensive. Additionally,absence of robust methods for selective RPE purification and removal of non-RPE cell impurities prevents upscaling of clinical quality RPE production. We aimed to address these challenges by developing a simplified hPSC-derived RPE production and purification system that yields high-quality RPE monolayers within 90 days. METHODS: Human pluripotent stem cells were differentiated into RPE using an innovative time and cost-effective protocol relying entirely on 2D cultures and minimal use of cytokines. Once RPE identity was obtained,cells were transferred onto permeable membranes to acquire mature RPE morphology. RPE differentiation was verified by electron microscopy,polarized VEGF expression,establishment of high transepithelial electrical resistance and photoreceptor phagocytosis assay. After 4 weeks on permeable membranes,RPE cell cultures were incubated with Dil-AcLDL (DiI-conjugated acetylated low-density lipoproteins) and subjected to fluorescence-activated cell sorting (FACS) for purification and subculture. RESULTS: Using our 2D cytokine scarce protocol,hPSC-derived functional RPE cells can be obtained within 2 months. Nevertheless,at this stage,most samples contain a percentage of non-RPE/early RPE progenitor cells that make them unsuitable for clinical application. We demonstrate that functional RPE cells express high levels of lipoprotein receptors and that this correlates with their ability to uptake lipoproteins. Combining photoreceptor uptake assay with lipoprotein uptake assay further confirms that only functional RPE cells uptake AcLDL. Incubation of mixed RPE/non-RPE cell cultures with fluorophore conjugated AcLDL and subsequent FACS-based isolation of labeled cells allows selective purification of mature functional RPE. When subcultured,DiI-AcLDL-labeled cells rapidly form pure homogenous high-quality RPE monolayers. CONCLUSIONS: Pure functional RPE monolayers can be derived from hPSC within 90 days using simplified 2D cultures in conjunction with our RPE PLUS protocol (RPE Purification by Lipoprotein Uptake-based Sorting). The simplicity of this protocol makes it scalable,and the rapidity of production and purification allows for high-quality RPE to be produced in a short span of time making them ideally suited for downstream clinical and in vitro applications. View Publication

The molecular basis of the earliest neuronal changes that lead to Alzheimer's disease (AD) is unclear. Here,we analyze neural cells derived from sporadic AD (SAD),APOE4 gene-edited and control induced pluripotent stem cells (iPSCs). We observe major differences in iPSC-derived neural progenitor (NP) cells and neurons in gene networks related to neuronal differentiation,neurogenesis,and synaptic transmission. The iPSC-derived neural cells from SAD patients exhibit accelerated neural differentiation and reduced progenitor cell renewal. Moreover,a similar phenotype appears in NP cells and cerebral organoids derived from APOE4 iPSCs. Impaired function of the transcriptional repressor REST is strongly implicated in the altered transcriptome and differentiation state. SAD and APOE4 expression result in reduced REST nuclear translocation and chromatin binding,and disruption of the nuclear lamina. Thus,dysregulation of neural gene networks may set in motion the pathologic cascade that leads to AD. View Publication

Upon immunogenic challenge,lymph nodes become mechanically stiff as immune cells activate and proliferate within their encapsulated environments,and with resolution,they reestablish a soft baseline state. Here we show that sensing these mechanical changes in the microenvironment requires the mechanosensor YAP. YAP is induced upon activation and suppresses metabolic reprogramming of effector T cells. Unlike in other cell types in which YAP promotes proliferation,YAP in T cells suppresses proliferation in a stiffness-dependent manner by directly restricting the translocation of NFAT1 into the nucleus. YAP slows T cell responses in systemic viral infections and retards effector T cells in autoimmune diabetes. Our work reveals a paradigm whereby tissue mechanics fine-tune adaptive immune responses in health and disease. View Publication

The formulations of peptide-based antitumour vaccines being tested in clinical studies are generally associated with weak potency. Here,we show that pharmacokinetically tuning the responses of peptide vaccines by fusing the peptide epitopes to carrier proteins optimizes vaccine immunogenicity in mice. In particular,we show in immunized mice that the carrier protein transthyretin simultaneously optimizes three factors: efficient antigen uptake in draining lymphatics from the site of injection,protection of antigen payloads from proteolytic degradation and reduction of antigen presentation in uninflamed distal lymphoid organs. Optimizing these factors increases vaccine immunogenicity by up to 90-fold and maximizes the responses to viral antigens,tumour-associated antigens,oncofetal antigens and shared neoantigens. Protein-peptide epitope fusions represent a facile and generalizable strategy for enhancing the T-cell responses elicited by subunit vaccines. View Publication